Despite advances in deciphering the molecular pathogenesis of acute myelogenous leukemia (AML), patients with relapsed/refractory (R/R) disease, particularly those with adverse genetic features (e.g., mutant p53 or FLT3-ITD) have grim prognoses, and effective therapies are lacking. Wee1, a critical cell cycle checkpoint kinase, has been identified as a target in AML by integrative genomic analysis; however, AZD1775, the first-in-class clinically relevant oral Wee1 inhibitor, has thus far been used primarily to potentiate genotoxic agent activity in p53-deficient solid tumors. Histone deacetylase inhibitors (HDACIs), epigenetic agents that induce tumor cell death through multiple mechanisms of action (MOAs), are approved for the treatment of CTCL/PTCL and have now been granted Orphan Drug status in AML. Recent attention has focused on novel MOAs of HDACIs involving disruption of the DNA damage response (DDR), including checkpoints and repair (e.g., HR and NHEJ). Our group discovered that AZD1775 interacts reciprocally and synergistically with pan- HDACIs (e.g., Belinostat) to kill human leukemia cells independently of p53 status, including those bearing FLT3-ITD. Of note, HDACI co-administration induced pronounced Wee1 and Chk1 inactivation, cdc2/Cdk1 dephosphorylation/activation at both Tyr15 and Thr14 sites, premature mitotic entry, and G1/S checkpoint abrogation, resulting in robust DNA damage (gamma-H2A.X) and apoptosis. Significantly, these interactions also occurred in primary AML cells harboring various NGS-defined mutations (e.g., p53, FLT3) and primitive AML progenitors, spared normal hematopoietic cells, and improved survival in vivo in mouse AML models. We now propose to translate these promising findings into the clinic through three Specific Aims. In Stage 1a(UH2a), we will validate in vivo MOAs for the AZD1775/ pan-HDACI Belinostat regimen e.g., Wee1 and Chk1 dephosphorylation/inactivation, cdc2/Cdk1 dephosphorylation/activation, and Bim, p-H3, and gamma-H2A.X up- regulation in AML xenograft models as candidate PD response determinants in subsequent trials in humans. In Stage #1b (UH2b), we will conduct a phase Ib trial of AZD1775/Belinostat in patients with R/R AML/MDS/CML-BC in order to identify the RPTD and characterize toxicities. The trial will also provide preliminary signals of activity and test the feasibility of NGS genomic profiling and PD/PK analysis as candidate biomarkers/response determinants. In Stage #2 (UH3), if indicated, we will conduct a two-stage phase IIa trial of the regimen to assess activity more definitively, and to determine whether specific genetic aberrations (e.g. in p53, FLT3) or correlative PD/PK studies can identify patients most likely to respond. Successful completion of these aims will introduce a novel and potentially effective Wee1/HDAC inhibitory strategy based on reciprocal disruption of the DDR, upon which leukemia cells with poor-prognostic mutations depend, into the therapeutic armamentarium for R/R AML, a disease for which satisfactory therapy is currently lacking. It may also provide a foundation for the more personalized use of this strategy in AML based on NGS/PD analysis.